Biological cell analyzing system

ABSTRACT

A biological cell analyzing system which is capable of automatically categorizing unstained biological cells as normal or non-normal. The cells are made to flow through a transparent tube in single file and are scanned with a mixture of ultraviolet and visible light. The cytoplasm and nucleus of each cell absorb ultra-violet radiation to different degrees, and the emergent light signal, as modulated by the scanned cells, is detected, amplified, and extended to a data processor which logically analyzes the signal from each cell on a real-time basis. The visible light signal is subtracted from the ultraviolet light signal to improve the signal/noise ratio of the latter, and to automatically cancel out non-biological debris. A number of acceptance tests are electronically performed on each cell, and if any of the tests is failed the cell is categorized as non-normal. Ambiguous conditions, resulting for example from the clumping of cells, are identified and separately counted. The system can process up to several thousand cells from a single sample during a one-minute run.

United States Paten 1 3,699,336 Ehrlich et al. 1 Oct. 17, 1972 Primary Examiner-Archie R. Borchelt BIOLOGICAL CELL ANALYZING SYSTEM Attorney-Amster and Rothstein [72] lnventors: Melvin P. Ehrlich, Roslyn Estates, A

L.I., N.Y.; Milton Stoller, West [57] ABSTRACT Hartford, Conn.; Stanley Grand, A biological cell analyzing system which is capable of Westbury; Robert De Cote, Flushautomatically categorizing unstained biological cells as ing, both of NY. normal or non-normal. The cells are made to flow through a transparent tube in single file and are [73] Asslgnee' Hycel Houston scanned with a mixture of ultra-violet and visible light. [22] Filed: Aug. 15, 1969 The cytoplasm and nucleus of each cell absorb ultraviolet radiation to different degrees, and the emergent L21] App! 850547 light signal, as modulated by the scanned cells, is detected, amplified, and extended'to a data processor 52 us. Cl ..2s0/s3.3 UV, 250/71 R, 356/39 which logically analyzes the signal from each cell on a 511 int. Cl. ..G0ln 33/16 real-time basis- The visible light Signal is subtracted [58] Field 6: Search ..250/83.3 uv,71 R; 356/39 from the ultra-violet light Signal to improve, the signal/noise ratio of the latter, and to automatically [56] References Cited cancel out non-biological debris. A number of acceptance tests are electronically performed on each UNlTED STATES PATENTS cell, and if any of the tests is failed the cell is 1 A 2,875,666 3/1959 Parker et al. ..356/39X categimed as mblgufms l 4 l 68 k 83 resulting for example from the clumping of cells, are 64 H/ 9 Kameilts 0/ UVX identified and separately counted. The system can 3497690 2/1970 f 250/83 3 UV X process up to several thousand cells from a single same a le durin a one-minute run. 3,523,756 8/1970 Loebl ..356/39X p g Claims, 34 Drawing Figures SCANNING AND 1w MONITORING SCANINER SYSTEM K QUARTZ W VISIBLE 97 /ucm AUTOMATIC 46 I VACUUM SAMPLE PUMP 8O HANDLINC I SYSTEM QUARTZ WASTE cm q v 32 RESERVOIR sflfltfigi," N3? mlsvstru [i J m0 FIO25 SCOPE FIGS.4'I

sa i #94 VIDEO CONOITIONER/ FICJT I32 81 O8 DATA 7) PROCESSOR F|GS.9-21

36) /T2 RECORDER AND PLAYBACK ELECTRONIC CELL SYSTEM SIMULATOR No.22 FIG-25 -61 40; 6A 42- l START/STOP, 2 COUNTER 1 DATA CONTROL WK Acoulsmou SYSTEM 57/ FIG. 24

PATENTEDDDT 11 I972 3.699.336

A 'SHEEI 01 F SCANNING AND FLYI MONITORING F G. POT

SCASNNER SYSTEM 84 48 uv AND QUARTZ 1 97 OPTICAL VISIBLE VACUUM SYSTEM /LIGHT GAUGE 3s AUTOMATIC 46; "-l I vAcuun SAMPLE PUMP HANDLING I SYSTEM DuARTz WASTECELL 'CAPILLARY T 32 RESERVOIR -54 "125,233?

TUBE LIGHT Box 44 SYSTEM 4 T TDD HG.25 SCOPE HGS.4-7

VIDEO CONDITIONER I32 DAN 50- 52 so,

DATA 12x88 4 l l PROCESSOR Z69 F|GS.921- T 56 H2 RECORDER AAD PLAYBACK x 1 ELECTRONIC CELL SYSTEM SIMULATOR H312 FIG.23.

START/STOP- 2 N 1 DATA CONTROL A A L ACQUISITION SYSTEM FIG. 24

ATTORNEYS INVENTORS PATENTEnnm 1 1 m2 SHEET 0211f 2S "VISIBLE BAND WAVELENGTH I" NILLINICRONS VISIBLE am WAVELENGTH m HILLIMICRONS PATENTED OCT 1 7 I972 SHEET 07 [1F 25 PATENTEDnm 17 1912 SHEET 08 0F 25 

1. A system for analyzing biological cells comprising a capillary tube, means for controlling a substantially laminar flow through said capillary tube of a carrier solution containing biological cells therein, means for irradiating said capillary tube with a source of movable focused light of wavelengths which are absorbed by said biological cells, and means for detecting the light transmitted through said capillary tube to determine characteristics of the biological cells flowing through said capillary tube.
 2. A system in accordance with claim 1 wherein the inner diameter of said capillary tube and the rate of flow of said carrier solution through said capillary tube are such that said biological cells are constrained to flow in essentially single file through said capillary tube.
 3. A system in accordance with claim 2 wherein a substantial portion of the energy of said light is contained in ultra-violet wavelengths lower than 300 millimicrons.
 4. A system in accordance with claim 1 wherein a substantial portion of the energy of said light is contained in ultra-violet wavelengths lower than 300 millimicrons and said light is irradiated on said capillary tube in the form of line sweeps across said tube.
 5. A system in accordance with claim 4 wherein said detecting means includes means responsive to the instantaneous intensity of the ultra-violet light at wavelengths Below 300 millimicrons transmitted through said capillary tube for deriving a signal proportional thereto and means for distinguishing between nuclear and cytoplasmic cellular material within said capillary tube along any line sweep across said tube in accordance with the instantaneous magnitude of the derived signal.
 6. A system in accordance with claim 5 wherein said capillary tube has an inner diameter and the rate of flow of said carrier solution through said capillary tube are such that said biological cells are controlled to flow in essentially single file through said capillary tube.
 7. A system in accordance with claim 6 wherein a portion of the total energy in said light is contained in wavelengths above 300 millimicrons, and said detecting means includes means for detecting the instantaneous intensity of the light at wavelengths above 300 millimicrons transmitted through said capillary tube and for generating a signal proportional thereto, and means for subtracting the generated signal from said derived signal such that the signal/noise ratio of the signal operated upon by said distinguishing means is increased.
 8. A system in accordance with claim 5 wherein a portion of the total energy in said light is contained in wavelengths above 300 millimicrons, and said detecting means includes means for detecting the instantaneous intensity of the light at wavelengths above 300 millimicrons transmitted through said capillary tube for generating a signal proportional thereto and means for comparing the instantaneous intensity of said generated signal to a threshold value to determine the presence of a histiocyte in said capillary tube.
 9. A system in accordance with claim 8 further including means for controlling a flow through said capillary tube of biological cells from different samples in succession, and means responsive to the determination of a number of histiocytes having flowed through said capillary tube which exceeds a predetermined number during the flow of cells from a particular sample for indicating that the sample contains lung cells.
 10. A system in accordance with claim 5 further including means for inserting said capillary tube into different containers in succession, each container containing sample cells from a different person, means for controlling the flow of cells from each sample through said capillary tube for a predetermined time interval prior to the change of samples, means for categorizing the cells flowing through said capillary tube in accordance with the operation of said detecting means, and means for delaying the categorizing of cells until cells from each sample have flowed through said capillary tube for a predetermined time interval and have cleaned the capillary tube of cells from the previous sample.
 11. A system in accordance with claim 3 wherein said capillary tube is made of quartz and said irradiating means includes quartz objective and condenser lenses disposed on opposite sides of said quartz capillary tube.
 12. A system in accordance with claim 11 further including a quartz plate for supporting said quartz capillary tube adjacent to said quartz objective lens and a droplet of oil bridging said quartz objective lens and said quartz capillary tube.
 13. A system for analyzing biological cells comprising a scanning station, means for controlling the movement of biological cells in essentially single file past said scanning station, means for scanning said cells at the scanning station with line scans of ultra-violet light having a substantial portion of its energy at wavelengths below 300 millimicrons, means for detecting the instantaneous intensity of the ultra-violet light transmitted through said cells at the scanning station for deriving a signal proportional thereto, means for distinguishing between nuclear and cytoplasmic cellular material along any line scan through a cell in accordance with the derived signal, and means responsive to said distinguishing Means for categorizing a cell in one of at least two groups.
 14. A system in accordance with claim 13 wherein said distinguishing means is operative to indicate the scanning of cytoplasmic cellular material responsive to the magnitude of said derived signal exceeding a first threshold level and is operative to indicate the scanning of nuclear cellular material responsive to the magnitude of said derived signal exceeding a second threshold level.
 15. A system in accordance with claim 14 further including means for maintaining said derived signal below said first threshold level at time periods corresponding to the leading and trailing portions of each of said line scans.
 16. A system in accordance with claim 14 further including means for characterizing each scanned cell as having one or more of a predetermined group of non-normal characteristics in accordance with the time intervals during each line scan through the cell when said derived signal exceeds said first and second threshold levels.
 17. A system in accordance with claim 16 further including means for deriving an annotation signal representative of the non-normal characteristics of each scanned cell, and means for combining said annotation signal and said derived signal in a form suitable for imaging the cell represented by said derived signal on a television display together with a code representative of its non-normal characteristics.
 18. A system in accordance with claim 14 further including means for deriving an annotation signal indicative of the groups in which each cell has been categorized, and means for combining said annotation signal and said derived signal in a form suitable for imaging the cell represented by said derived signal on a television display together with a code representative of the groups in which said cell has been categorized.
 19. A system in accordance with claim 13 further including means for scanning said cells with line scans of light having a portion of its energy at wavelengths above 300 millimicrons, means for detecting the instantaneous intensity of the light at wavelengths above 300 millimicrons transmitted through said cells for generating a signal proportional thereto, means for comparing the instantaneous magnitude of said generated signal to a threshold level, and means responsive to the instantaneous magnitude of said generated signal exceeding said threshold level for registering that the cell being scanned is a histiocyte.
 20. A system in accordance with claim 13 wherein said scanning station includes a quartz capillary tube through which said cells move, and quartz objective and condenser lenses disposed on opposite sides of said quartz capillary tube.
 21. A system in accordance with claim 20 further including a quartz plate for supporting said quartz capillary tube adjacent to said quartz objective lens, and a droplet of oil bridging said quartz objective lens and said quartz capillary tube.
 22. A system in accordance with claim 13 wherein a portion of the total energy in said light is contained in wavelengths above 300 millimicrons, and said detecting means includes means for detecting the instantaneous intensity of the light at wavelengths above 300 millimicrons transmitted through said cells for generating a signal proportional thereto, and means for subtracting the generated signal from said derived signal such that the signal/noise ratio of the signal operated upon by said distinguishing means is increased.
 23. In a system for scanning biological cells at a scanning station the improvement comprising a capillary tube disposed at said scanning station, and means for controlling a flow through said capillary tube of a carrier solution containing therein the biological cells to be scanned, said capillary tube having an inner diameter and the rate of flow of said carrier solution being such that said biological cells flow in essentially single file through said capillary tube wherein said cells are scanned by a source of movable ultra-violet light and said capillary tube is made of quartz, said system further including quartz objective and condenser lenses disposed on opposite sides of said quartz capillary tube.
 24. A system in accordance with claim 23 further including a quartz plate for supporting said quartz capillary tube adjacent said quartz objective lens, and a droplet of oil bridging said quartz objective lens and said quartz capillary tube.
 25. A system for line scanning biological cells comprising means for irradiating said biological cells with ultra-violet light of wavelengths below 300 millimicrons in the form of line scans, means for deriving a signal dependent upon the ultra-violet light absorption characteristics of a scanned cell, means for simultaneously irradiating said biological cells with light of wavelengths above 300 millimicrons in the form of lines scans, means for generating a signal dependent upon the above 300 millimicron light absorption characteristics of a scanned cell, and means for processing the derived signal in accordance with the generated signal to increase the signal/noise ratio of said derived signal.
 26. A system in accordance with claim 25 wherein the nucleus and cytoplasm of the biological cells which are scanned absorb above 300-millimicron and below 300-millimicron light to different degrees, and further including means for distinguishing between nuclear and cytoplasmic regions of a cell in accordance with said derived signal exceeding first and second threshold levels, said processing means including means for subtracting said generated signal from said derived signal to facilitate the distinguishability of nuclear and cytoplasmic cell regions.
 27. A system for detecting histiocytes comprising means for controlling the line scanning of biological cells with light of wavelengths above 300 millimicrons, means for generating a signal dependent upon the light absorption characteristics of a scanned cell, and means responsive to the generated signal exceeding a predetermined threshold level for characterizing a scanned cell as a histiocyte.
 28. A system in accordance with claim 27 wherein said line scanning controlling means includes a capillary tube and means for controlling a flow through said capillary tube of a carrier solution containing biological cells therein, said capillary tube having an inner diameter and the rate of flow of said carrier solution through said capillary tube being such that biological cells are controlled to flow in essentially single file, the flow rate of said biological cells through said capillary tube and the speed of said line scanning being such that said cells are scanned along lines approximately perpendicular to the direction of cell movement.
 29. A system for controlling line scans with focused ultra-violet light of flowing biological cells comprising a capillary tube, means for controlling a flow through said capillary tube of a carrier solution containing biological cells therein, means for generating successive ultra-violet light scans, an objective lens disposed on one side of said capillary tube for coupling said ultra-violet light to said capillary tube, a condenser lens disposed on the opposite side of said capillary tube, and means for generating an electrical signal in accordance with the ultra-violet light transmitted through said condenser lens.
 30. A system in accordance with claim 29 further including a plate for supporting said capillary tube adjacent to said objective lens and a droplet of oil bridging said objective lens and said capillary tube. 